The processes of nucleic acid (DNA) amplification and subsequent nucleic acid probe assay are well known and have been implemented in a variety of formats. While these formats are highly effective, they are somewhat difficult to perform in the clinical laboratory. Generally, DNA amplification and assay reactions are performed sequentially on the sample to be assayed; that is, the DNA amplification reaction is first carried out to completion, and the DNA probe assay is then performed on the fully amplified sample. This is referred to as an end point assay.
One problem with end point assays is that the amplified DNA (amplicons) from the DNA amplification reaction must be physically transferred to the subsequent DNA probe assay. Because of the transfer, the potential exists for contaminating the laboratory environment with the DNA amplicons. In addition, the general risk of misidentifying a given sample or coiinfusing it with other samples increases each time that a physical transfer of the sample takes place.
There have been previous proposals for self-contained test units that are capable of carrying out an integrated nucleic acid ampliffcation and nucleic acid assay on a liquid biological sample while the sample remains confined within the test unit. For example, U.S. Pat. No. 5,229,297, to Paul N. Schnipelsky et al, describes a cuvette for DNA amplification and detection which comprises a plurality of flexible compartments for containing a sample, ampliiying reagents and detection reagents, together with passageways connecting the sample and reagent compartments with a detection site and waste compartment. A roller is used to squeeze or compress the sample and reagent compartments in a desired sequence, thereby forcing the sample and detection reagents through the passageways to the detection site and waste compartment. Teraporary seals are used to isolate the sample and reagent compartments from the passageways until sufficient pressure is generated by the roller. Although this arrangement is advantageous in that the sample remains within the cuvette during amplification and detection, the need for a roller to break the temporary seals and cause the various fluids to flow between compartments introduces undesirable complexity and makes it difficult to automate the amplification and assay procedure.
In U.S. patent application Ser. No. 08/277,553, U.S. Pat. No. 5,639,428 fled by Hugh V. Cottingham on Jul. 19, 1994, an improved test unit for carrying out integrated nucleic acid amplifications and nucleic acid assays is disclosed. In the improved test unit, the flow of sample and reagent liquids is controlled by centrifugal force applied by a relatively simple rotating apparatus, thereby avoiding the need for rollers and other complex mechanisms. While this represents a substantial improvement over the arrangement disclosed in U.S. Pat. No. 5,229,297, the need to provide for controlled fluid movement within the test unit still exists and renders the test unit somewhat more complex than might be desired.
In addition to the end point assays discussed previously, homogenous methods of nucleic acid assay also exist. Homogeneous methods do not require the physical transfer of the amplified material to a separate assay site, but rather function simultaneously with the amplification reaction. Examples of known homogenous assay methods include fluorescence polarization, fluorescence energy transfer and light absorbance. While fluorescence polarization, in particular, functions very well in a research laboratory, it has a significant drawback in that it requires glass sample tubes or cells. This is a result of the fact that most plastic processing methods, such as injection molding or thermoforming, create stresses in the material of the finished part. These stresses have random polarization effects, and interfere with the transmission of polarized light that is required for a fluorescence polaization assay.
As is well known, DNA amplification reactions must occur within a certain temperature range in order to produce the desired number of amplicons. If the sample and the DNA amplification reagents are allowed to react before the sample reaches the required temperature, a phenomenon known as "mis-priming" can occur. This can affect the validity of the assay results, both in the case of an end point assay and a homogeneous assay.
In view of the foregoing, a need exists for a device or a test unit which is capable of carrying out an integrated DNA amplification and DNA probe assay with minimal complexity, and preferably without requiring fluid movements to occur within the test unit itself. There is also a need for a test unit which can be used to carry out a homogenous DNA probe assay using fluorescence polarization methods, but which does not require the use of glass to properly transmit polarized light. Finally, there exists a need for a test unit which can be used to carry out an integrated DNA amplification and DNA probe assay in a simple and effective manner, while preventing inadvertent mis-priming of the amplification reaction. The present invention is directed to fulfilling these objectives.
It is an object of the present invention to provide a DNVA amplification and homogenous DNA probe assay device in a "card" format that can be conveniently handled by clinical laboratory personnel, and accommodated in a suitable test apparatus.
It is another object of the invention to provide a unitary DNA amplification and DNA probe assay device which includes a multiplicity of sample cells, with each sample cell comprising the element and reagents needed for a DNA amplification reaction and a homogeneous DNA probe assay.
It is a further object of the invention to provide a unitary DNA amplification and DNA probe assay device in which all reagents needed for both DNA amplification and DNA probe assay are contained, in dried form, within the device, so that the addition of a liquid biological sample is all that is needed to carry out the amplification and assay procedure.
It is a further object of the invention to provide a test unit and method that performs a "hot start" of the DNA amplification reaction, thereby avoiding an invalid assay result due to mis-priming of the amplification reaction.
It is a further object of the invention to provide a test unit which has the optical properties necessary for a fluorescence polarization assay, but which can be made of inexpensive plastic materials rather than glass.
It is a further object of the invention to provide a test unit and method that yields instantaneous DNA probe assay data by means of a kinetic or dynamic measurement of DNA amplicons, rather than a conventional end point measurement.
It is a further object of the invention to provide a fluorescence polarzation DNA probe assay device which includes an integral polarizer, allowing for the use of a confocal polarization method.
It is a further object of the invention to provide an integral DNA amplification and homogenous DNA probe assay device that can be permanently sealed after the introduction of a liquid biological sample, thereby preventing amplicon contamination of the laboratory environment.
It is a still further object of the invention to provide an integrated DNA amplification and DNA probe assay device which can accommodate a plurality of liquid biological samples in discrete sample cells, and which can provide DNA probe assay data in a matter of minutes.